The endoplasmic reticulum (ER) is a folding factory for the cell, able to churn out thousands of secretory proteins per second. Misfolding of a number of these proteins results in diseases ranging from cystic fibrosis to liver cirrhosis. While full-length proteins can be studied in vitro using a myriad of biophysical techniques, thus allowing biophysicists to characterize in detail their folding energy landscapes, studying protein folding in the more physiologically relevant ER environment presents daunting technical obstacles. Using recently developed, powerful single molecule fluorescence techniques on nascent polypeptide chains that contain fluorescently-labeled amino acids, we will determine the conformational evolution as a nascent polypeptide chain elongates in the ER and how conformational space is modulated by interactions with the chaperones and modifying enzymes resident in the ER. The goal is to obtain detailed information on the folding landscape of the growing nascent chain, rivaling the data available in test-tube experiments. Co- and post-translational interactions between the ER-resident proteins and nascent chains likely smooth the folding energy landscape, biasing against misfolded and aggregation-prone intermediates. This remodeling of the folding landscape is particularly important for proteins with high contact order, i.e. many contacts between distal parts of the chain. We will focus on an important family of secreted proteins, the inhibitory serpins, which fold into native structures with high contact order. Intriguingly serpin native states are energetically metastable, poised to change to a different structure. Serpins play crucial biological roles by regulating proteases involved in key physiological processes including blood coagulation and inflammation. A number of serpin mutations, associated with diseases such as liver cirrhosis and emphysema (together called 'serpinopathies'), lead to serpin aggregation in the ER. By studying serpin folding in the ER and comparing it to in vitro folding, we will determine how the folding landscape is altered by interactions with ER-resident proteins and by disease-associated mutations. The results of this research will provide unprecedented insight into how serpins misfold and how their energy landscapes differ in vitro and in vivo. This work will also provide a readily accessible experimental toolbox for scientists studying protein folding in the ER as well as a platform for potential therapeutic strategies to treat serpinopathies and other ER folding diseases. PUBLIC HEALTH RELEVANCE: Secretory proteins, many of which are associated with diseases, begin life in the endoplasmic reticulum (ER). By deploying powerful biophysical and cell biological approaches, we will explore the folding energy landscape in the ER of a biomedically significant secreted protein from the serpin family and determine how it is altered by disease-causing mutations.